The three encephalitic alphaviruses, Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV) are endemic throughout North, Central and South America. Horses and humans are normally exposed after being bitten by infectious mosquitoes and the infection can develop into encephalitis with high rates of morbidity and mortality. These viruses are potential biological threat agents, being highly infectious through the aerosol route of exposure, easily produced in large amounts, and relatively stable in the environment. Currently, there is no licensed vaccine to the three encephalitic alphaviruses, and efforts to move vaccine candidates forward into clinical trials have not been successful. In this report, the development of recombinant modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-based vaccines for the three encephalitic alphaviruses is described. The recombinant vaccines MVA-BN-VEEV, MVA-BN-WEEV, and MVA-BN-EEEV were constructed and produced. The protective efficacy of these vaccines was evaluated in vivo. Female Balb/c mice were immunized subcutaneously with 108 tissue culture infective dose 50 (TCID50) of single MVA-BN encephalitic alphavirus vaccine or a triple mixture of three vaccines at a four-week interval. The mice were then instilled intranasally with 1×103 to 1×104 plaque forming units of VEEV, WEEV, or EEEV 14 days after the second immunization. The M